Patent Application
20210371823
The present disclosure relates to a method for expanding human dendritic cells (DC), and, especially, a method for expanding DC cells by using a viral transactivator protein from simian T cell lymphotropic virus (STLV). The disclosure also relates to a human DC cell and data repository constructed by the method.
Cancer is the number one killer of all mankind and the biggest threat to human health and longevity. China has the largest number of cancer deaths and the fastest increase in tumor incidence in the world. A relevant literature (CA Cancer J. Clin. 2016; 66(2): 115-32 PMID: 26808342) shows that the number of cancer cases in China in 2015 was 4.29 million, and the death toll was as high as 2.81 million. Immunotherapy is a revolutionary new technology after surgery, radiotherapy, and chemotherapy, and it has shown great potential in anti-cancer therapy. The concept of restoring or enhancing T cell function to achieve tumor killing has been validated. Antibodies targeting immune checkpoints, such as PD1, PDL1, and CTLA4, are effective in clinical therapy for certain types of human cancer. The genetically engineered CAR-T and TCR-T cells have shown amazing anti-cancer effects in certain kinds of cancer, particularly in chemo-resistant leukemia. However, it has been recognized that therapies with immune checkpoint antibodies, CAR-T and other cell technologies, remain to be modified for improved safety, efficacy and versatility. Immune tolerance and immune escape remain big concerns in cancer therapy.
Immunotherapy is not only aimed at cancer research, but it also plays a key role in diseases caused by pathogenic viruses. It is well known that, after a virus infects the body, it attacks host cells in an acute or chronic manner, causing inflammation, tissue and organ damage, and even life-threatening consequences in some cases. Some viruses, such as SARS coronavirus, influenza virus, Ebola virus, Zika and dengue fever virus, etc., spread rapidly after infecting the host. Some viral infections are associated with very high mortality, and there are no effective preventive or therapeutic drugs for these viral infections. In addition, there are some chronic viral infections, such as hepatitis B virus (HBV), HIV (Human Immunodeficiency Virus, HIV), human papillomavirus (HPV), Epstein-Barr virus (EBV), etc. Long-term infection by these viruses could possibly lead to various types of cancer, and currently there is a lack of curative therapeutic regime. Therefore, it is imperative to find an immunotherapeutic method that can combat deadly viral infections.
The immune system is the most important host defense system for recognizing and removing foreign pathogenic microorganisms (such as viruses) and mutated cells (such as tumors) in the body, and the full display of its functions and activities is the key to controlling cancer progression and the aforementioned viral infections. In the immune system, the functional units that play a role are various types of immune cells, including macrophages, B lymphocytes, T lymphocytes, natural killer cells (NK), DC cells, etc. Among them, T lymphocytes and NK cells mainly mediate cellular immunity, while B lymphocytes prevent the invasion of foreign antigens by producing neutralizing antibodies and mainly participate in humoral immunity. DC cells are the most capable professional antigen presentation cells (APC), participating in both the cellular immunity and humoral immunity. DC cells are the controller of the entire immune system, bridging innate immunity and adaptive immune responses in protecting host from invading pathogens or cancerous transformation of somatic cells.
It is well known that DC cells function as professional APCs, and one of their primary functions is to capture antigens, process antigenic peptides followed by presenting them to reactive naive T lymphocytes, thereby promoting T cell activation, proliferation and differentiation into antigen-specific, effector cytotoxic T lymphocytes (CTL) as well as long-lived memory T cells. Antigen-specific CTLs have a direct killing effect on relevant cancer cells and virus-infected cells in an HLA-restricted manner. Moreover, DC cells have the capacity to activate NK cells. DCs can promote the proliferation of NK cells and enhance the activity of NK cells by expressing a variety of membrane proteins and secreting cytokines such as IL12, IL15, TNFalpha and interferon gamma. In addition, studies have found that certain cytokines can induce B cells to produce IgG and IgA antibodies, and DC cells can increase the secretion of IgG and IgA after contacting with B cells. DC cells can migrate to the germinal centers of lymph nodes after loaded with antigens, interact with B cells there, and regulate B cell humoral immunity. In conclusion, DC cells regulate the host immune response through a variety of mechanisms and are the one of the most valuable cell types in immunotherapy.
DC-based immunotherapy has been extensively evaluated in clinical trials. These clinical studies provide two aspects of information. First, DC cells are safe to use as cancer therapeutic vaccines. Second, DC cancer vaccines demonstrated therapeutic efficacies on certain types of human cancer in clinical trials, however, the effectiveness of DC-based immunotherapy demands further improvement. The he current conventional methods for DC preparation suffer several technical deficiencies. First, primary blood DC cells are extremely rare, and there is no technique currently available to efficiently expand primary DCs in culture for cancer patients. Therefore, it is a huge difficulty to achieve a sufficient DC cell number that is suitable for patient treatment. Second, the currently utilized DC cells in therapy are derived from blood monocytes in which these monocyte-derived DCs or MoDCs are generated by a complex maturation and activation process involving the use of multiple cytokines and stimuli. Recent studies argue that MoDCs are phenotypically distinct from blood DCs, which may explain undesirable therapeutic efficacies in clinical trials. Third, the MoDC preparation method still requires at least 100 ml blood for manufacturing a single dose of DC cells for infusing into patients for therapy. Besides, the suboptimal activity of MoDCs, poor antigen loading on DCs and inefficient homing capacity to lymphoid tissues of MoDCs are additional deficiencies that are associated with the current conventional DC method. It is obvious that these technical hurdles in DC methods limit clinical applications of DCs as the most powerful antigen-presenting cells in mediating host protective immunity against cancer and viral diseases that are resistant to conventional chemotherapies.
In order to overcome the above-mentioned problems, in one aspect, the present disclosure provides a method for expanding DC cells ex vivo, which comprises contacting a cell sample containing DC cells to be expanded with a viral transactivator protein or Tax derived from a simian virus called simian T lymphotropic virus (STLV), which is known to be non-pathogenic to humans.
In some embodiments, the contacting comprises introducing an expression vector of the STLV Tax protein into the DC cells to be expanded and allowing the STLV Tax protein to be expressed in the DC cells to be expanded.
In some embodiments, the introducing is performed by lentiviral transduction.
In some embodiments, the STLV virus is selected from the group consisting of STLV1, STLV2, STLV3, and STLV4 viruses.
In some embodiments, the Tax protein comprises the amino acid sequence as set forth in SEQ ID NO: 1, 2, 3 or 4.
In some embodiments, the cell sample is peripheral blood or cord blood.
In some embodiments, the DC cells to be expanded are obtained by the following steps: a) isolating mononuclear cells from the cell sample; and b) inducing the mononuclear cells to differentiate into DC cells.
In some embodiments, step b) comprises contacting the mononuclear cells with PHA and IL-2 sequentially or simultaneously, or contacting the mononuclear cells with GM-CSF and IL-4.
In some embodiments, the method further comprises introducing an expression vector of a tumor-associated antigen, a tumor-specific antigen or a viral antigen into the expanded DC cells.
In some embodiments, the tumor-associated antigen is hTERT.
In some embodiments, the method further comprises continuing to culture the expanded DC cells in a medium containing IL2 for 3 weeks to 2 months.
In some embodiments, the method does not comprise the step of isolating the DC cells to be expanded from the cell sample.
In some embodiments, the expansion success rate of the method is over 80%.
In another aspect, the present disclosure provides DC cells obtained by the method described above.
In some embodiments, the DC cells are CD11c+ and CD205+.
In some embodiments, the DC cells are CCR7+, HLA-DR+, and CD83, and/or CD40+, CD70+, 4-1BBL+, CD80+, CD83+, CD86+.
In another aspect, the present disclosure provides a method for preparing a cell culture with tumor cell or virus-infected cell killing activity from peripheral blood or cord blood mononuclear cells, which comprises allowing the peripheral blood or cord blood mononuclear cells to be co-cultured with DC cells prepared by the expansion method described above.
In another aspect, the present disclosure provides a cell culture prepared by the method described above.
In some embodiments, the cell culture comprises CTL cells, NK cells, and NKT cells.
In some embodiments, the CTL cells comprise Tα/β cells and Tγ/δ cells.
In another aspect, the present disclosure provides use of the cell culture described above in the preparation of anti-tumor or anti-viral drugs.
In another aspect, the present disclosure provides a method for preparing CTL cells from peripheral blood or cord blood mononuclear cells, which comprises allowing the peripheral blood or cord blood mononuclear cells to be co-cultured with DC cells prepared by the expansion method described above, and isolating CD3+ cells from the co-cultured cells.
In some embodiments, the CTL cells comprise Tα/β cells and Tγ/δ cells.
In another aspect, the present disclosure provides a method for preparing NKT cells from peripheral blood or cord blood mononuclear cells, which comprises allowing the peripheral blood or cord blood mononuclear cells to be co-cultured with DC cells prepared by the expansion method described above, and isolating CD3+ and CD56+ cells from the co-cultured cells.
In another aspect, the present disclosure provides a method for preparing NK cells from peripheral blood or cord blood mononuclear cells, comprising allowing the peripheral blood or cord blood mononuclear cells to be co-cultured with DC cells prepared by the expansion method described above, and isolating CD3- and CD56+ cells from the co-cultured cells.
In another aspect, the present disclosure provides a method for establishing a DC cell and data repository, which comprises respectively obtaining expanded DC cells corresponding to each subject from cell samples of multiple subjects by the expansion method described above.
In some embodiments, the number of subjects is greater than 400.
In some embodiments, the method further comprises detecting and recording the surface marker molecules of the expanded DC cells, the activity of the expanded DC cells, and HLA type of the expanded DC cells.
In some embodiments, the detection of the activity is performed by co-cultivating the expanded DC cells and peripheral blood mononuclear cells, and detecting the killing activity of the obtained cells.
In some embodiments, the method further comprises cryopreserving the expanded DC cells corresponding to each subject separately.
In some embodiments, the method further comprises periodically detecting and recording changes in the activity of the cryopreserved DC cells.
In another aspect, the present disclosure provides a DC cell and data repository prepared by the method described above.
In another aspect, the present disclosure provides a method for preparing a cell culture with tumor or virus killing activity from peripheral blood or cord blood mononuclear cells of a subject, which comprises:
The DC cell expansion method provided by the present disclosure can efficiently and highly selectively expand DC cells from peripheral blood or cord blood mononuclear cells, and the expanded DC cells have the ability to induce the generation of CTL cells, NK cells and NKT cells from peripheral blood or cord blood mononuclear cells. The human DC cell resource bank provided by the present disclosure can provide highly active DC cell lines for autologous or allogeneic use, which is beneficial for the treatment of tumors or viral infections.
Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.
“DC cells to be expanded” refers to an object to be expanded by the method of the present disclosure, and it may include immature DC cells and mature DC cells. “DC cells to be expanded” may be purified DC cells or DC cells in peripheral blood or cord blood. That is, it is not necessary to separate DC cells from other cells (such as T cells, B cells) before expansion.
“Simian STLV viruses” refer to the form of primate T-cell lymphotropic virus (PTLV) in apes, and are currently only found in a few monkeys and gorillas. In addition to simian viruses (STLVs), PTLV viruses also include human viruses (HTLVs). Both human HTLV viruses and simian STLV viruses have multiple subtypes. Among human HTLV viruses, HTLV1 and HTLV2 are more common. Approximately 5% of people infected with HTLV1 will get sick, including human T-cell leukemia/lymphoma and tropical spastic paralysis. HTLV2 virus infection has little relevance to diseases. The relationship between HTLV3 and HTLV4 virus infection and disease is not yet known. Among simian STLV viruses, 1% of apes infected with STLV1 virus have been found to develop a simian leukemia. The remaining STLV2, STLV3, and STLV4 viruses have not been reported to cause simian diseases, and there is no evidence that they are related to human diseases.
“Tax protein” refers to a protein encoded by the simian STLV virus, which has a domain capable of activating the NF-κB, AP1 and/or STAT signaling pathways, and can promote the expression of viral genes, the transcription of host cellular genes and the transformation of host cells. The inventors unexpectedly discovered that the Tax protein of the simian STLV virus can selectively promote the proliferation and activation of DC cells in mononuclear cells derived from peripheral blood or cord blood, with a success rate of over 80%. In some embodiments of the present disclosure, the Tax protein includes the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4.
“Tumor-associated antigen” refers to an antigen that is abnormally highly expressed in tumor cells and has a rather low expression level in normal tissue cells, such as human telomerase reverse transcriptase (hTERT), tumor-testis antigen (CTA), such as MAGE family protein, NY-ESO-1, gp100, GPC3, AFP protein, etc. “Tumor-specific antigen” refers to a protein with amino acid change(s) due to gene mutation(s) in tumor cells, such as hRAS, kRAS, etc.
“HLA typing” refers to the identification of the major histocompatibility complex (MEW) phenotype of a subject or its cells. Methods of HLA typing include traditional serological methods and cytological methods, as well as DNA typing methods developed in recent years, such as PCR-RFLP, PCR-SSO, and so on. Commonly used typing loci include HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, HLA-DQ, etc. A “match” of HLA type herein refers to the identity of the alleles of typing loci, involving a match of HLA types between DC cells and peripheral blood mononuclear cells (PBMC) to be co-cultured with the DC cells to induce the production of a cell populations containing CTL cells and a match of HLA types between CTL cells and the cells to be killed by them (such as tumor cells).
A “DC cell and data repository” refers to a cell bank that includes multiple (for example, more than 400, more than 1,000, more than 10,000, or even more than 100,000) separately stored DC cell lines. These cell lines usually originate from healthy individuals. These cell lines can be obtained from the mononuclear cells of the peripheral blood (or cord blood) of healthy individuals using the method of the present disclosure. The method for expanding DC cells provided in the present disclosure can selectively and efficiently expand DC cells from the mononuclear cells of each individual, which is very beneficial to the establishment of such DC cell and data repository. In addition to storing these cell lines, the cell and data repository also keeps other information about each cell lines, such as HLA types, DC cell activity, surface markers, quality control data, preservation records, and so on. The DC cell and data repository of the present disclosure is particularly useful for providing HLA-matched, activated DC cells for allogeneic subjects, which is conducive to solving the clinical problem of HLA matching.
A “subject” refers to a healthy individual, an individual suffering from or suspected of suffering from a certain disease (preferably a human). The term can generally include “healthy volunteer,” “patient,” “test object,” and “treatment object,” etc.
In some embodiments, the present disclosure provides a method for expanding DC cells, which involves the use of a Tax protein from a simian STLV virus. As mentioned above, the present inventors discovered that the expression of the STLV Tax protein in a DC cell is able to promote the expansion and activation of the DC cell. The Tax protein may be from STLV1, STLV2, STLV3, or STLV4 virus, and accordingly, may have an amino acid sequence as set forth in SEQ ID NO: 1, 2, 3, or 4. It should be pointed out that, the polypeptides obtained by conservatively substituting one or several amino acids in these sequences, or deleting one or several amino acid residues at the amino or carboxyl end may still have similar functions, so they should also be encompassed within the scope of the present disclosure. The DC cells to be expanded may be primary DC cells in PBMCs, or immature DC cells and mature DC cells (such as monocyte-derived dendritic cells, MoDC) formed by inducing monocytes in PBMCs. Due to the high selectivity of the method provided in the present disclosure, it is generally unnecessary to perform a DC cell separation step with a cell sample containing the DC cells before the expansion. Coding nucleotides of the STLV Tax protein can be introduced into DC cells in various ways to achieve the expression of the STLV Tax protein in the DC cells. These introduction methods include, for example, liposome- and other positively charged transfection reagent-mediated transfection, electrotransfection, adenovirus or retrovirus-mediated transfection, and the like. In the Examples of the present disclosure, a method for introducing the coding nucleotide sequence of the STLV Tax by lentiviral transduction is provided (see below).
In addition to the introduction of the Tax encoding gene into the expanded DC cells, coding nucleotide sequences of other target proteins can also be introduced, such as tumor-associated antigens or tumor-specific antigens or viral antigens (such as HBV antigens HBsAg, HBcAg, HBxAg, etc.) HCV antigens NS1, NS2, NS3, NS4, NS5, etc.; HPV antigens such as E6, E7, etc.; EBV antigens such as EBNA1, LAMP2, etc.; HTLV virus antigens such as Tax, HBZ, etc.; HIV virus antigens such as Gag, etc.; conserved core antigens of SARS, Ebola, influenza viruses, etc.). DC cells with these antigenic proteins can prime naive T cells to differentiate into CTL cells that are specific for tumor cells or virus-infected cells expressing these target proteins, thereby mediating their antigen-specific killing on target cells.
In some embodiments, the present disclosure provides a method for preparing a cell culture having tumor cell or virus-infected cell killing activity from peripheral blood or cord blood mononuclear cells. The method comprises performing a mixed cell culture of the peripheral blood or cord blood mononuclear cells and the above described DC cells carrying corresponding target proteins (tumor antigens or virus antigens). The cell culture produced form the mixed culture comprises not only CTL cells (including Tα/β cells and Tγ/δ cells), but also NK cells and NKT (natural killer T) cells, which is able to kill tumor cells or virus-infected cells by MHC-restricted and non-MHC-restricted mechanisms. When applied to patients, different mechanisms can act synergistically and it will have a better therapeutic effect.
In some embodiments, the present disclosure provides a method for preparing a DC cell and data repository, which comprises separately obtaining expanded DC cells (DC cell lines corresponding to each subject from cell samples of a plurality of subjects (usually healthy individuals) by using the DC cell expansion method disclosed herein.
In some embodiments, the present disclosure provides a method for preparing a cell culture with tumor or virus killing activity from peripheral blood mononuclear cells of a subject (usually a tumor patient or a virus-infected patient), which comprises 1) performing a HLA typing of the subject's peripheral blood mononuclear cells; 2) selecting DC cells which are HLA-matched with the subject's peripheral blood mononuclear cell from the DC cell and data repository provided in the present disclosure; and 3) performing a mixed culture with the subject's peripheral blood mononuclear cells and the DC cells. The DC cell and data repository of the present disclosure provides a great convenience for the cell HLA matching in this method.
Particular embodiments of the present disclosure will be further described in detail below in conjunction with specific operation steps.
Construction of Virus Vector that Expresses STLV Tax Protein and Production of Lentivirus
Coding nucleotide sequences of the STLV Tax genes were introduced into DC cells through lentiviral vectors and expressed in the DC cells.
The Tax genes from four STLV viruses were cloned into lentiviral vectors. The amino acid sequence corresponding to the Tax genes were shown in SEQ ID NO: 1 to 4. Synthesis of the nucleotide sequences of the Tax genes were entrusted to a third-party service platform. The nucleotide sequences were inserted into lentiviral vectors, bacterial competent cells were transformed, and after confirmation through sequencing, plasmids were extracted and purified with a plasmid purification kit to obtain high-quality plasmids of the recombinant expression vectors, i.e., STLV Tax plasmids.
293 cells (or 293T cells or 293FT cells) were co-transfected with the obtained STLV Tax plasmids and the packaging plasmids through transfection reagents (such as calcium phosphate transfection reagent, liposome transfection reagent, high molecular polymer transfection reagent, etc.). The packaging plasmids include expression plasmids of VSV-G, Gag-Pol and Rev.
The viruses (hereinafter referred to as STLV Tax lentivirus) could be collected by two centrifugation methods. Method 1: Collect the virus supernatant and centrifuge at 25,000 rpm at 4° C. for 2 hours. Method 2: Add PEG8000 and appropriate concentration of NaCl solution to the virus supernatant, and centrifuge at 1600 g or 3000 rpm for 30-60 min at room temperature or 4° C. After centrifugation, the supernatant was discarded, the virus pellet was resuspended in RPMI1640 medium or other liquid suitable for virus preservation, and then frozen in a deep-low temperature refrigerator (−80° C.). The MOI value was measured before storage or before use.
A tumor or virus antigen gene was cloned into a suitable lentiviral vector to produce a corresponding lentivirus by using a method similar to that for the preparation of the STLV Tax lentiviruses described above.
10 mL of peripheral blood (or cord blood) collected from a healthy person or a patient was placed in an anticoagulation tube and sent to a cGMP-level laboratory for mononuclear cell isolation. Here, a density gradient centrifugation method or any known separation methods could be used to separate the mononuclear cells. General steps of density gradient centrifugation for the separation of mononuclear cells were as follows:
DC cells could be cultured and expanded from the isolated peripheral blood or cord blood mononuclear cells in two ways.
Method 1: expansion of mature and highly active DC cells through primary dendritic cells. The general steps were as follows:
As an alternative to steps 1) to 3), peripheral blood or cord blood mononuclear cells could also be co-stimulated and cultured in a medium containing PHA and IL2, and then processed with the subsequent steps.
Method 2: Expansion of mature and highly active DC cells through monocyte-derived dendritic cells (MoDC). The general steps were as follows:
8) After the cells were continued to be cultured in a medium containing 50˜200 units/mL IL2 for about 3 weeks to 2 months, the expression of CD83, CD80, CD86, CD70, CCR7, 4-1BBL, HLA-DR and the like was analyzed by FACS, confirming that a large number of mature and highly active dendritic cells were obtained.
The steps are briefly described as follows:
Referring to
Active DC cells in the HDCR are co-cultivated with PBMCs (or cord blood mononuclear cells) from tumor or virus-infected patients or healthy volunteers to perform a mixed lymphocyte reaction to produce CTL and NK cells.
The steps are briefly described as follows:
The steps are briefly described as follows:
We enriched and attained a large number of cells from each collection of mononuclear cells through the methods described above, including preparation of lentivirus containing STLV Tax gene, isolation of mononuclear cells, preliminary stimulation (cytokine induction) and lentiviral transduction, subsequent culture and screening processes. These cells were subjected to immunophenotyping analysis by flow cytometry, and it was found that they expressed DC cell-related marker molecules.
We obtained 6 collections of peripheral blood from healthy adults through volunteer donation, each with 7 to 9 mL peripheral blood. The time period from blood collection to the start of blood immune cells harvesting and processing was controlled within 3 hours. The 6 blood samples with different individuals were subjected to PBMC separation by the method for separating PBMCs described above. After the cells of buffy coat were obtained, they were washed with PBS, resuspended in a serum-containing medium, and PHA factor was added to stimulate and culture the cells. Starting on the second day, 200 units/mL (final concentration) of IL2 protein was added to the cells for culture. After 3 days of stimulation and culture, the stimulated cells were transfected with the STLV3 Tax lentivirus, and IL2 was added to continue the culture. Afterwards, change or add IL2-containing medium according to the cell growth status and continue the culture. After 3-4 weeks of culture, some cells would grow slowly and die gradually, and the remaining cells would continue to be cultured. After two months of continuous culture, some cells for flow cytometry analysis and morphological observation (as described in Example 1) were collected, which confirmed that the cells that continued to grow were DC cells. Among the 6 peripheral blood samples, one sample gradually died after being cultured for a period of time, while the remaining cells of 5 samples were cultured for more than 4 months. After culturing for 2 months and confirming that they were DC cells through flow cytometry, cryopreserving some of the continuously expanding cells was begun, and then continued to culture the remaining cells. Later, the cryopreserved cells were resuscitated and then cultured, and it was found that they still maintained good growth and expansion capabilities. Therefore, the method of the present disclosure successfully expanded active DC cells from 5 out of 6 peripheral blood samples (83% success rate). It was decided that the expansion failure of the other one of the samples might be caused by an operational error. To confirm this hypothesis, the donor of the blood sample that failed to expand was found and asked for another donation of 8 mL of peripheral blood. The method of the present disclosure was used to carry out the culture and expansion again, and after a culture period of at least 4 months or more, the expansion and activation were successful. Therefore, technically speaking, the DC cell expansion method provided by the present disclosure can efficiently achieve the expansion and activation of DC cells, and the success rate is close to 100%.
On the other hand, it is known that DC cell account for only 1% of the number of PBMC cells, T cells account for about 58%, B cells account for about 20%, NK cells and monocytes account for about 10%, and NKT cells account for about 1%.
On the other hand, it is known that DC cell account for only 1% of the number of PBMC cells, T cells account for about 58%, B cells account for about 20%, NK cells and monocytes each account for about 10%, and NKT cells account for about 1%. Only 7-9 mL of peripheral blood was obtained from healthy volunteers. After separation, the obtained 0.8-1.5×107 PBMC cells were all used for the expansion of dendritic cells. During the culture, there would be two groups of cells: cells of one group were easy to aggregate into a clump to grow, and cells of the other group were individually scattered in the medium. It was found by continuous observation that after 3 to 5 weeks of culture, the number of cells growing in clumps would increase, while individual scattered cells gradually disappeared. Therefore, after culturing for 2 months according to the method of the present disclosure, the cells obtained were basically clumped cells. Without any sorting treatment, it was proved by various marker molecules and morphology that these cells were pure DC cells. This is especially important for allogeneic use to prevent other potentially contaminated cells, such as T cells, from causing graft-versus-host disease.
Telomeres in normal cells progressively shorten as cells divide. When they reach a certain level, the cells will age and die. Activation of the telomerase is able to keep the lengths of telomeres relatively stable, thereby enabling cells to gain the ability to proliferate indefinitely and further develop carcinogenesis. Therefore, the abnormality of telomerase activity is closely related to the occurrence and development of tumors. Telomerase is composed of telomerase reverse transcriptase (hTERT), telomerase RNA and a pseudouridine synthase, and hTERT is a restrictive component of the telomerase activity. Studies have found that hTERT is highly expressed in more than 90% of all types of tumor and plays an important role in tumorigenesis and cancer development. Because it is almost not expressed in most normal tissue cells, hTERT is a very ideal tumor target.
Based on this, in this example, a DC cell line (BJ8, established after transfection with STLV4 Tax protein, HLA phenotypes A0201+ and A2402+) was selected from the DC cell and data repository, and transduced with lentivirus to continuously express hTERT (DC-hTERT). PBMCs, which were HLA-A matched with the DC-hTERT cells, were selected and subject to MLR test to co-culture and stimulate the production of a large number of immune cells. The latter was sorted by CD3 magnetic beads to obtain a separate group of cells. This group of T cells were mainly CD8+ T cells, as observed by flow cytometry (
In this example, HLA-A0201+/A2402+ PBMC cells were co-cultivated with the parental DC cells and the DC-hTERT of Example 3 to produce TERT-specific T cells. These two types of T cells were tested for their killing effect on tumor cells after sorted out by CD3 magnetic beads.
First, this study needed to confirm that the T cell killing was hTERT specific. Therefore, in this study, human osteosarcoma tumor cell U2OS (HLA-A2) was choose as target cells. A major feature of this target cells was that the background expression of TERT protein was very low, almost undetectable. U2OS/TERT modified by genetic engineering could express hTERT protein. The study selected the parental U2OS and U2OS/TERT as target cells to detect the killing effect of hTERT-specific T cells. It could be seen from
Next, three melanoma cell lines were used as target cells, and normal human fibroblast NHF (HLA-A2/A2) was used as a control (
In another study, three cell lines of NL20 cells (bronchial epidermal cells that could proliferate infinitely after genetic modification, HLA-A1/3), H1299 (lung cancer cells, HLA-A32/A24) and normal human fibroblasts (NHF, HLA-A2/A2) were choose as research objects. First, the three cell lines were genetically modified to allow NL20 cells express HLA-A0201 molecules, which could be recognized by T cells with the same HLA type; to allow H1299 cells express HLA-A0201 molecules, so that the cells were matched with T cells with the same HLA-type (both HLA-A0201 and HLA-A2402 were all matched); to allow NHF cells express the cancer testis antigen MAGEA3, and whether hTERT-specific T cells could kill the cells was observed. From the results shown in
One of the dilemmas faced by tumor immunotherapy is tumor immune escape. Due to the heterogeneous characteristics of tumor tissues, tumor cells in the same tissue may exhibit different immune characteristics, and the level of MHC expression may be high or low, or even not expressed at all. In this case, a single treatment with HLA-restricted killer T cells has some effects in the short term, but in the long term, it may not prevent the escape of residual immune cells, leading to tumor recurrence. Therefore, a coordinated treatment with immune cells with different killing mechanisms is a trend in future immunotherapy. A major feature of the DC cells in the DC cell and data repository provided by the present disclosure is that they can stimulate the activation and proliferation of CTL cells, and can also induce a certain proportion (25% to 90%) of CD56+ cells, i.e., NK Cells and CD3+CD56+ NKT-like cells. Using this group of mixed immune cells for tumor treatment can achieve a synergistic effect and prevent immune escape.
In this example, three DC cell lines were selected from the DC cell and data repository, and they were mixed and cultured with HLA-matched PBMCs:
A DC cell line code-named BJ3 (established after transduction with STLV3 Tax) was mixed with a collection of PBMC cells, both of which belonged to the HLA-A2 phenotype.
A DC cell line code-named BJ19 (established after transduction with STLV3 Tax) was mixed with a collection of PBMC cells, both of which belonged to the HLA-A2 phenotype.
A DC cell line code-named BJ8 (established after transfection with STLV4 Tax) was mixed with a collection of PBMC cells, both of which belonged to the HLA-A0201 and HLA-A2402 phenotypes.
A FACS analysis was performed on the cell cultures obtained after culture, and the results were shown in
A DC cell line (BJ9) in the DC cell and data repository was used to induce PBMC expansion to produce two populations of CD3+ and CD3−CD56+ cells with the method described herein. After a negative selection with CD3 magnetic bead, a large number of CD3−CD56+ cells were obtained, i.e., NK cells (
It should be pointed out that some of the examples provided herein were performed using Tax proteins from STLV3 and STLV4 viruses, but similar results were obtained using Tax proteins from viruses of other subtypes (STLV1, STLV2) (not shown).
It can be seen from the above description that the DC cell expansion method provided in the present disclosure can achieve: in vitro large-scale culture of primary DC cells in a cGMP environment, breaking through the number limit for therapy; ensuring that all DC cells are in a highly activated state; ensuring that DC cells are ready for genetical modification to efficiently and stably present any tumor or viral antigens as needed, achieving the characteristics of high antigen load rate and multiple indication; effectively expanding DC cells from any individual, that is, a high efficiency of DC cell line establishment; effectively expanding a large number of DC cells via collecting only a small amount of peripheral blood or cord blood (≤10 mL); the expanded DC cells can be permanently cryopreserved and remain their activity after resuscitation.
In terms of safety, the possibility of Tax protein of ape STLV causing human diseases has not been reported, so the risk of cross infection is extremely low. In addition, the STLV Tax protein is a foreign protein to the human body and has high immunogenicity. It can also play a function similar to that of an immune adjuvant, stimulating the activity of human immune cells and strengthening anti-tumor or anti-viral effects.
Therefore, based on the method of the present disclosure, a human DC cell and data repository is provided, that is, through the DC cell expansion method provided in the present disclosure, DC cells derived from healthy population or early-stage cancer population are engineered to realize their proliferation potential and function; at the same time, data information of each collection of DC cells (including HLA typing, molecular expression profile related to DC cell maturation and activation, in vivo and in vitro activity, etc.) are obtained. The human DC cell and data repository has great application value. First of all, it can achieve the goal of strengthening the function of autologous DC cells and serve the future treatment or prevention for cancer or viral diseases. For example, the DC cell and data repository accepts blood samples from healthy population or early-stage cancer patients with non-transmittable diseases, from which DC cells are extracted and activated, and stored in a deep low temperature environment after passing the quality inspection. When it is needed to use autologous DC cells for disease or prevention purposes in the future, the DC cell and data repository can provide a sufficient number of DC cells with potent activity. Secondly, allogeneic use of DC cells and resource sharing are realized in a way of HLA matching. All DC cells in the DC cell and data repository are screened by standard procedure and do not carry any transmittable pathogens. When cancer or acute virus infection patients cannot use their own immune cells due to low function, after HLA matching, a DC cell line suitable for the patient can be quickly selected in the DC cell and data repository. The DC cell line can be thawed out, and applied to patients very quickly to save precious time for them. Third, the DC cell and data repository can provide DC cell resources for the national and even global public health and medical technology fields. It is a strategic resources for studying and conquering certain known or unknown acute viral infectious diseases.
Tax protein amino acid sequences of STLV viruses:
| Number | Date | Country | Kind |
|---|---|---|---|
| 201810368646.3 | Apr 2018 | CN | national |
This application is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/CN2019/075529, filed Feb. 20, 2019, designating the United States of America and published as International Patent Publication WO 2019/205783 A1 on Oct. 31, 2019, which claims the benefit under Article 8 of the Patent Cooperation Treaty to Chinese Patent Application Serial No. 201810368646.3, filed on Apr. 23, 2018, entitled “A Method and Application for Expanding Mature and Highly Active Human Dendritic Cells In Vitro.”
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/075529 | 2/20/2019 | WO | 00 |
